The aim of the proposed research is to determine the passive ion permeability and regulation of volume of isolated serotonin granules as well as the intact platelet, and the effect which ion gradients and induced ion fluxes (particularly that of calcium) have upon the mechanisms of serotonin homeostasis. Particular emphasis will be placed upon the delineation of the region, regulation, bioenergetics, and physiological role of the proton gradient across the serotonin granule membrane, especially with regard to the effect upon serotonin distribution across the serotonin granule membrane and the integrity of serotonin stores. The techniques are available and will be further developed for preparation of the highest purity of isolated serotonin granules, platelet plasma membranes, and intact platelets. Collaboration with other investigators will allow the obtaining of platelets and serotonin granules which have been artificially enhanced or depleted of critical membrane components, as well as those from patients with constitutional disorders of platelet function. The approach will involve spectrophotometric, potentiometric, and radiochemical techniques which have been applied with great success to other organelle systems in the measurement of the passive ion permeability, internal pH, and regulation of volume. Ion specific metallochronic indicators and dual wavelength spectroscopy, atomic absorption spectroscopy, and electron probe analysis in intact platelets and isolated organelles will be used to quantitate the compartmentalization of ions, particularly Ca ions, within the platelet. New nuclear magnetic resonance techniques will be employed which will allow the measurement of the pH and ATP concentration of the cytosol and serotonin granules within the intact platelet. The knowledge of physico-chemical properties of the intact platelet and isolated granules gained from these experiments will be correlated with the effect of the variations of these parameters on serotonin uptake, storage, and release. These conclusions, combined with the pathological correlation gained from platelets with altered function, will form the basis for the understanding of serotonin homeostasis within the platelet and platelet function as it relates to serotonin uptake, storage, and release.